Choline supplementation: Impact on broiler chicken performance, steatosis, and economic viability from from 1 to 42 days

This study was carried out to compare the impact of choline supplementation (available from two sources synthetic and natural) on various dosages in broilers. The mode of choline supplementation, via diet and additional sources, synthetic and natural, and the data of performance, carcass quality, blood parameters, and hepatic steatosis were compared. A total of 1050 day-old male Cobb 500 broiler chicks were randomly assigned to 10 treatments, using a completely randomized design model in a factorial scheme, with 6 replicates per treatment and 25 birds per replicate. Choline was supplemented using three sources: synthetic choline chloride 60% (CC), and two sources of natural choline A (NCA), and B (NCB). The Control treatment did not receive any choline supplementation. The diets were supplemented with low, intermediate and high doses of choline sources (400g/t, 800g/t, and 1200g/t of CC; 100g/t, 200g/t, and 300g/t of both NCA and NCB). Data analysis was performed using a factorial model to investigate the effects of choline supplementation (CC, NCA, NCB) and doses on the measured variables. Overall, the results indicated that the the performance of NCA was better than CC & NCB, specifically the dose of 100g/t of NCA outperformed MAR at 100g/t & CC at 400g/t, leading to a significant increase in body weight gain (85.66g & 168.84g respectively), and a noteworthy (9- & 12-point respectively) improvement in feed conversion ratio. Furthermore, NCA contributed to a reduction in steatosis when contrasted with various NCB & CC doses, likely due to the presence of curcumins and catechins in the natural choline source. These findings demonstrated that NCA supplementation yielded superior results compared to CC and NCB across both performance and liver health aspects in broilers aged 1 to 42 days. In conclusion, NCA can be used to replace the CC 60% without compromise on the zootechnical performance in broilers.

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Introduction
The use of choline in commercial broiler feed is an important aspect in the feed industry to enhances the growth performance and regulates the lipid metabolism as well.Typically, choline plays a significant role in many important metabolic pathways, being crucial for the structural maintenance of cell membranes and organelles, acting as a constituent of phospholipids (Zeisel, 1991;Marimuthu et al. 2019); synthesis of methionine from homocysteine as a labile methyl donor; and a precursor molecule for the formation of acetylcholine in the nervous system.In addition, the choline is a component of very-lowdensity lipoproteins (VLDL) which involves in triglyceride transport out of the liver and thereby reduces the accumulation of fat in the liver (D'souza et al. 2022; Khose et al. 2019;Selvam et al. 2018;Xu et al. 2010).It also inhibits the fatty acid synthesis by downregulating fatty acid synthase gene expression as well as attenuating its activity (Sherriff JL et al. 2016;Zhu J et al. 2014).Furthermore, choline is referred to as a "lipotropic" factor due to its role in increasing fat utilization, which results in the reduction of fat deposition in the body (Farina et al. 2017;Selvam et al. 2018).
However, the absence of choline, or levels below the recommendation, increases deleterious effects on meat chickens, such as reduction in growth and perosis, especially in younger birds (Selvam et al. 2018;Santiago, et al. 2020).Choline supplementation is commonly done by synthetic sources, but high hygroscopicity and oxidation with loss of vitamins, its corrosive nature, as well as the trimethylamine formation in the intestinal tract of broilers (the common reason less than half of the choline chloride is absorbed), allowed th te scientist to look for an alternative (Zeisel et al. 1986;Calderano et al., 2015).
In this respect, research has been developed to unveil potential substitutes for synthetic sources, and formulations with herbs have been gaining ground with consistent results in hepatoprotective capacity and improvement of performance parameters.These natural products, produced from selected plants and blends of herbs, which can mimic the function of choline (Calderano et al. 2015).Chandrasekaran et. al (2019) when investigating the lipotropic activity of herbal formulations containing Acacia nilotica and Curcuma longa, demonstrated the lipotropic effect of this natural source which was highlighted by low fat accumulation and anti-lipogenic activity.The formulations of natural choline using herbs such as Acacia nilotica and Curcuma longa paved a way to get the associating benefits and thus enhancing the effects of hepatoprotective action which was being reported discretely (Yarry et al. 2009, Narayanan et al. 2013).Hence the current study was carried out to compare the impact of choline supplementation (available from two sources synthetic source and natural sources) on various dosages in broilers.The mode of choline supplementation, via diet and additional sources, synthetic and natural, and the data of performance, carcass quality, blood parameters, and hepatic steatosis were compared.

Housing conditions and management
Housing, feeding regimens, and rearing conditions used at the Federal University of Paraiba (UFPB) are thought to be representative of modern commercial broiler operations in Brazil.This trial was carried out at an experimental farm located in Areia -Paraiba, Brazil.Farm facilities comprise 88 identical floor pens.Of these, 42 pens were used.The trial was carried out with 1050 day-old male Cobb 500 broiler chicks, organized in a factorial experiment, 3 x 3 + 1. Chicks were randomly assigned to 1 of 10 treatments, with 6 replicates per treatment and 25 birds per replicate.Individual pens measuring 1.5 x 1.5 m served as experimental units.Pens were consecutively numbered with the respective treatment number using pen cards.
Birds were kept in appropriate (i.e., optimum) environmental conditions (temperature) for age during the trial.Lighting was provided using fluorescent bulbs.The experimental period totaled 42 days.A 3-phase feeding program was used as follows: starter (days 1-21), grower (days 22-35), and finisher (days 36-42).Feed was provided in a pan feeder (capacity, 20 kg).Water was offered ad libitum using drinkers.
Standard floor pen management practices were used throughout the experimental period.Birds and facilities were inspected twice daily.The following data were recorded: general health status of broilers, environmental temperature, and constant feed and water supply.Dead birds were removed, and unexpected events were identified.Deaths, including pen number, date of death, bird weight, and potential diagnosis, were recorded using a Daily Mortality Record.

Animals
Day-old male Cobb chicks hatched from eggs produced by 30 weeks-old breeders and weighing 45g on average were obtained from Frango Dourado hatchery (Pernambuco, Brazil).Twenty-five male broiler chicks were housed in each pen.Birds were sexed at the hatchery.

Dietary treatments
Choline was supplemented using three sources: synthetic choline chloride 60% (CC), and two sources of natural choline A (NCA), and B (NCB).The Control treatment did not receive any choline supplementation.The diets were supplemented with low, intermediate and high doses of choline sources (400g/t, 800g/t, and 1200g/t of CC; 100g/t, 200g/t, and 300g/t of both NCA and NCB).Natural Choline A (NCA) is indexed as Kolin Plus (M/s Natural Remedies Pvt Ltd, Bengaluru, India), is a polyherbal formulation containing a combination of Acacia nilotica (A.nilotica) and Curcuma longa (C.longa) belonging to the families of Mimosaceae and Zingiberaceae, respectively.Natural Choline B (NCB) contains mainly Achyranthes aspera, Trachyspermum ammi, Azadirachta indica, and others.

Feed
Mash feeds were manufactured at UFPB Agrarian Center -Campus II, located in Areia, Paraiba, Brazil.Broiler diets were formulated with feedstuffs widely used in Brazil.Diets were representative of local commercial feed formulations and designed to meet or exceeded nutritional requirements for broiler strain and age.Feed batches were independently mixed and bagged.Feed bags were labeled with a trial number, mixing date, type of feed, dietary treatment, and replicate number.Detailed records of feed mixtures and test product inventories were kept.Test feeds were mixed for 4 minutes at 28 rev/min in a 100 kg capacity mixer.The experimental diets were described in Table 1 -

Statistical analysis
The statistical analysis of the characteristics studied was performed using the R software, version 4.2.1 (2022).The analysis of variance (ANOVA) assumptions (error normality, random and independent errors, and variance homoscedasticity) was met.
Data analysis was performed using a factorial model to investigate the effects of choline supplementation (CC, NCA, NCB) and doses on the measured variables.A two-way analysis of variance (ANOVA) was conducted to assess the main effects of choline supply and doses, as well as any interactions between them.The significance level was set at α=0.05 for all tests.Post hoc Tukey tests were conducted to compare the means of each choline source and dose to identify specific differences.

Results
There was a significant interaction observed in body weight (BW) (P=0.0405),body weight gain (BWG) (P=0.0394), and feed intake (FI) (P=0.0139), as shown in Table 4.In the overall effect, birds exhibited higher weights with CC supplementation compared to the Without group and NCB (P=0.033), but similar to NCA (P>0.05).Weight gain significantly improved with the intermediate dosage compared to the control (P=0.164),but was similar to the low and high dosages (P>0.05).The BWG data were consistent with the findings regarding final body weight in all aspects.There was no significant effect observed on feed consumption, feed conversion ratio, and survival rate.Higher body weight (BW) was observed with choline supplementation via CC at the intermediate dosage (P=0.034) and high dosage (P=0.0032), while no significant difference was found at the low dosage (P=0.1232), as evidenced by the data presented in Table 5. Notably, there was no observable effect on the incremental doses with CC supplementation (P=0.0948) or NCA supplementation (P=0.7947).However, NCB supplementation resulted in a significantly higher final weight at the intermediate dosage compared to the low and high dosages (P=0.0150).The BWG data exhibited similar patterns to those observed in BW.Regarding feed intake, significantly higher values were obtained with NCB supplementation compared to NCA (P=0.0161),although no significant difference was observed between NCB and CC (P>0.05).Moreover, NCB showed greater feed intake at the intermediate dosage compared to the high dosage (P=0.0029), while no significant difference was found between the intermediate and low dosages (P>0.05).There was a significant interaction observed in BW (P=0.0164),BWG (P=0.0164),FI (P=0.0339), and FCR (P=0.0097), as presented in Table 6.In the overall effect, feed conversion ratio (FCR) showed higher values with NCB supplementation compared to NCA (P=0.008) but was similar to CC and the Without supplementation (P>0.05).7. The BWG data showed similar effects to those obtained in BW.NCB exhibited higher feed intake at the intermediate dosage compared to the high dosage (P=0.0039)but was similar to the low dosage (P>0.05).The FCR demonstrated better results with NCA compared to CC and NCB at the low dosage (P=0.0425).At the intermediate dosage, the FCR showed higher values with the NCB source compared to the CC and NCA sources (P=0.003).The FCR with the use of CC (P=0.2294) or NCA (P=0.5825) is similar to the evaluated dosages, whereas, with NCB, there is a decrease in effectiveness at the intermediate dosage compared to the other dosages of this additive (P=0.0026).There was a significant interaction observed in BW (P=0.0166),BWG (P=0.0167), and FCR (P=0.0457), as shown in Table 8.In the overall effect, FCR showed better results with NCA compared to NCB and CC (P=0.0432), and was similar to the Without supplementation (P>0.05).9.The CC supplementation resulted in higher BW with intermediate and high doses compared to the low dose (P=0.326), while NCA showed better results with the low dose compared to the others (P=0.0203).The NCB was not influenced by the doses (P=0.6306).Low dose weight gain was higher with NCA than with CC (P=0.0445), but similar to NCB.FCR showed better results with NCA compared to NCB (P=0.0405), but similar to CC.
NCA demonstrates similarity to NCB, however, FCR significantly differs between them in the 1-42 day phase (P=0.0405).The similarity of NCA with CC in this variable indicates that they are equivalent in providing choline satisfactorily.However, NCA shows better results with doses of 100g/t to 200g/t, while CC shows better results with doses of 800g/t to 1200g/t.The breast yield was lower when NCB was used (P=0.0036),as shown in Table 10.On the other hand, this source promoted higher thigh yield (P=0.0251).The leg yield was better with the low dose of choline supplementation (P=0.0065).The breast yield was influenced by the supplementation of the high dosage, with a superiority (P=0.0002) of CC and NCA sources compared to NCB, Table 11.Regarding thighdrums yield, there was a better performance with NCB compared to the others (P=0.0383).NCB performed better with the low dosage than the intermediate (P=0.0017)but was similar to the high in ThighDrums yield.A higher cholesterol content was obtained with the control group compared to the intermediate (P=0.0591), while it was similar in the low and high choline supplementation dosages, as shown in Table 12.Meanwhile, the low dosage promoted a higher liver lipid content compared to the other dosages (P=0.0394).In this context, the supplemented choline sources reduced the steatosis score compared to the diet without choline supplementation (P<0.001), with a reduction observed in all three evaluated dosages compared to the control (diet origin; P<0.001)In the interaction, steatosis showed that it is reduced with the supplementation of an additional source in the diet.However, better results were obtained with NCA and NCB compared to CC (P<0.001), as shown in Table 13.NCA showed a better result than NCB and CC even at the low dosage (P<0.001),consistently achieving better results at the intermediate and high dosages (P<0.001).A lower score was observed at the high dosage of NCA (P<0.001).The liver triglyceride content was higher with CC supplementation compared to NCA (P<0.001), but similar to NCB (P>0.05) at the intermediate dosage.Price estimates based on values listed in feed Tables, July 2022.Combined analysis of liveability (i.e., the final number of birds per treatment), performance, carcass yield, and economic data, which can be adjusted according to natural variations, revealed relative gross margin fluctuations across different scenarios (Table 14).
Using to the Control treatment as a reference, the relative gross income derived from breast, thigh, drumstick, and whole chicken sales is 100%.The same applies to the remaining treatments in the same order.Hence, 100 is a used benchmark to estimate the delta between treatments.
Based on comparative analysis between the control and remaining treatments, NCA fed at 100 g/t yields the highest relative gross income (107.42,i.e., 7.42 p.p higher the than control).Margins derived from NCA at 100 g/ton are also higher relative to the three different doses of Choline Chloride or the NCB product (400 g/t Choline Chloride vs. 100 g/t NCA; 10.54 points; 800 g/t Choline Chloride vs. 200 g/t NCA, 4.06 p.p.; 1,200 g/t Choline Chloride vs. 300 g/t NCA, 4.40 points).NCA fed at 200 g/t, or 300 g/t outperformed the control and Choline Chloride fed at three different doses.
The NCB product was economically superior to the control treatment when fed at 100 g/t and 300 g/t doses, However, 0.6 p.p. losses were estimated at the 200 g/t dose.The NCB product outperformed Choline Chloride fed at 400g/t.In contrast, when fed at 200 g/t, the economic performance of the NCB product was poorer (4 to 5 points less) compared to Choline Chloride fed at 800 g/t or 1,200 g/t (Table 10; highlights in red).Assessment estimates suggest NCA was more efficient than the control treatment, Choline Chloride or NCB at doses used in this trial.

Discussion
During the total period of this trial (1-42 days), statistical differences were observed for the performance variables evaluated by Tukey analysis.Furthermore, employing orthogonal contrasts, it was evident that NCA exhibited superior performance compared to the NCB product.Specifically, NCA demonstrated notable enhancements in terms of body weight (BW) with an increment of 61.54 g/bird (P = 0.0143), body weight gain (BWG) with an increment of 61.59 g/bird (P = 0.0142), and feed conversion ratio (FCR) with an improvement of 7.6 points (P = 0.0190).Furthermore, birds fed with NCA displayed a trend of lower feed intake by 114.4 g/bird, although this difference was not statistically significant.This aligns with a study by Khose et 2021) investigated the effects of replacing synthetic choline with a natural choline source and demonstrated no significative difference on zootechnical performance and nutrition utilization and carcass characteristics.However, the current results clearly demonstrates the importance of choline supplementation and additionally, the results demonstrate the superiority of natural sources over synthetic ones, with significant results, especially with NCA.
Exploring carcass characteristics, the study revealed that at 200g/t, NCA led to an increase in leg weight, while at 300g/t, it caused a decrease in thigh weight and an increase in breast weight compared to NCB at 300g/ton.Additionally, NCA at 200g/ton exhibited a higher carcass yield compared to Choline chloride at 800g/t.These findings were corroborated by Khose et al. (2019), which demonstrated the advantages of herbal choline supplementation at 0.5 kg/ton of feed in terms of enhanced liver protection, carcass traits, and economic viability in broiler production.Moreover, a comparative analysis was conducted among NCA, choline chloride, and NCB.Significant distinctions emerged between NCA versus Choline chloride and NCA versus NCB in terms of controlling fatty liver.NCA exhibited greater efficacy in mitigating fatty liver incidence in broilers compared to choline chloride and NCB, showing a dose-dependent effect.In this study, NCA at 200 g/t or 300 g/t outperformed the Control and Choline Chloride at various doses.In a similar vein, Gangane et al. (2010) reported reduced fatty liver incidence in broilers through herbal choline feeding.NCA contains certain compounds with proven hepatoprotective effects, such as polyphenols & curcuminoids.Therefore, findings from this trial, particularly the significant reduction in fatty liver development, may be attributed to curcumin and catechin contained in the natural source of choline employed.Studies have shown that curcumin supplementation can improve the growth performance of broiler chickens (Zhang et al., 2017;Upadhaya et al., 2016;Huang et al., 2015;Zhang et al., 2018).Moreover, the phytoconstituents (polyphenols and curcuminoids) present in NCA modulates the liver genes accounts for fat metabolism/catabolism and lipogenesis (Marimuthu et al., 2022).This might enhance the energy availability at the muscular level which inturn enhanced fat utilization.Because of that, the NCA outperformed CC and NCB, as shown by better animal performance parameters and best carcass yields.
As anticipated before, combined analysis of performance data and economic estimates 257 suggest a beneficial effect of NCA compared to remaining treatments, and that NCA given at 258 the 100 g/t dose yields the highest relative gross margin.Economic assessment findings 259 estimate to support the superiority of NCA relative to NCB and CC in this trial in terms of 260 animal performance as well as carcass and cut yield in broilers aged 1 to 42 days, particularly 261 when fed at 100 g/t.In this study's conditions, the dosage of 100g/ton probably was enough to 262 attend to the broilers' requirements due to the low oxidative stress conditions and the use of a 263 corn-soy-based diet.In field conditions with challenges such as low quality of ingredients, 264 heat stress, and high density of animals, higher doses of the NCA evaluated in this study 265 (200g/t or 300g/t) can be recommended.From a practical standpoint (i.e., product storage and 266 feed logistics) the lower dose requirement is another advantage of NCA relative to Choline 267 Chloride, the commercial doses recommended of CC (400, 800, or 1,200 g/t) can involve 268 several space-related and logistics concerns.Therefore, the incorporation of NCA at a dosage 269 of 100g/t in broiler feed could serve as a viable substitute for choline chloride and NCB.270 271 272

Conclusion 273
Natural Choline A is a more effective source of supplemental choline relative to the 274 other sources evaluated.Natural Choline A is a safe source of choline with enhanced 275 hepatoprotective effects, which ensures better animal performance and carcass quality.Natural 276 Choline A also has greater economic efficiency compared to Chloride Choline and Natural 277 Choline B. The dose of 100 g/t of Natural Choline A is recommended in low oxidative stress 278 conditions.In higher exposure to heat stress, it is recommended doses of 200g/ton or 300g/t 279 of Natural Choline A in the diet to broilers from 1 to 42 days of age.280 Introduction adjust L1: "diets" instead of "feed" L2: "contribute to" instead of "grow and in" L18: "…its activity (Sherriff et al. 2016;Zhu et al. 2014)."L21-22: Please delete "published in the Journal of Nutrition" L23-24: Please delete "published in the American Journal of Clinical Nutrition" L27: Please delete "A study published in the Journal of Nutrition found that" L30: "The absence or deficiency of choline at under the recommended levels increases…" L32: "provided" instead of "done" L35: "allows us to observe"?L46: "increases the reduction"?Housing conditions and management adjust L4: "1050-day old male" or "1500 day-old male"?Animals adjust L2: 39g or 45 g (as in abstract)?"Diets" instead of "Feed" adjust L8: "are presented" instead of "were described" L35: "to estimate the delta"?adjust L39: "…using the Folch method (1957), and lipids…" L57: "in lower BW and less BWG" compared to?L73: "…of Choline Chloride vs 300 g/t of the MAR product…" L79: Are you sure?Same superscripts L88: "…and FCR (improvement by 8 points; P = 0.0015) (Table 5)."L89: "KPFC fed at 100 g/t improved FCR by 8 points" compared to?L93: Please rephrase "effects did not differ significantly between treatments" L161-166: Please remove this part to discussion L226: ZEISEL or ZEIZEL?Reviewer #2: General: The objective of the experiment reported is to determine the impact of using three sources of choline chloride, each at three levels, and study effect in broilers using various responses.I must acknowledge the authors' hard work in carrying out the experiment and following up with statistical analysis.Nevertheless, the manuscript has too many weaknesses, as pointed out below.The authors must first remember to put line numbers in their manuscripts.That will make the life of the reviewer much easier.The language used in the manuscript makes it difficult to follow the science.It distracts from the message and needs to be completely overhauled.The statistical analysis is inadequate and incorrectly done, considering how the treatments were arranged (see details below).The writing of the manuscript is rather untidy, making it look like it was haphazardly put together.The authors must clearly explain the choice of the levels of each choline chloride source they used.
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al. (2018) that suggested herbal choline supplementation at 0.350 and 0.500 kg/ton of feed can effectively substitute synthetic choline chloride-60%, typically used at 1 kg/ton of broiler feed.Similarly, Calderano et al. (2015) evaluated the use of a vegetal source of choline in five levels of supplementation as a replacement of choline chloride at 60% and did not find any difference between the sources on the performance parameters, but they concluded that the use of 100 mg.kg-1 of a vegetal source of choline to replace the use of choline chloride in corn-soybean meal diets for broilers from 1 to 42 days of age.Likewise, Kathirvelan et al. (2013) reported comparable body weight outcomes for both natural and synthetic choline-supplemented groups.Correspondingly, Selvam et al. (2018) demonstrated that the NCA at 400 g/ton replaced the function of 1 kg/ton of synthetic choline (choline chloride 60%) in broilers, showing the potential to mimic biological activities of synthetic choline through the restoration of negative effects caused by a choline-deficient diet.On the other hand, Zhang et al. (

Table 4 : Effect of choline supplementation on zootechnical parameters of broilers at 21 days of age
IBW -Initial body weight; BW -Body weight; BWG -Body weight gain; FI -Feed intake; FCR -Feed conversion ratio; LIVE -Liveability; 1 Dosage means the dose of each choline source, being Low (100g/t for NCA and NCB and 400g/t for CC), Intermediate (200g/t for NCA and NCB and 800g/t for CC), and High (300g/t for NCA and NCB and 1200g/t for CC).a,b Means followed by different letters in the same column differ significantly Tukey test, P<0.05.

Table 5 : Effect of interaction between the supplementation sources of broilers at 21 days of age
Body weight gain; FI -Feed intake; a,b Means followed by different letters in the same line differ significantly Tukey test, P<0.05.A, B Means followed by different letters in the same column differ significantly Tukey test, P<0.05.

Table 6 : Effect of choline supplementation on zootechnical parameters of broilers at 35 days of age
Higher BW was observed with choline supplementation via CC at intermediate and high dosages compared to low dosages (P=0.099), as indicated by the data presented in Table IBW -Initial body weight; BW -Body weight; BWG -Body weight gain; FI -Feed intake; FCR -Feed conversion ratio; LIVE -Liveability; 1 Dosage means the dose of each choline source, being Low (100g/t for NCA and NCB and 400g/t for CC), Intermediate (200g/t for NCA and NCB and 800g/t for CC), and High (300g/t for NCA and NCB and 1200g/t for CC.a,b Means followed by different letters in the same column differ significantly Tukey test, P<0.05.

Table 7 : Effect of interaction between the supplementation sources of broilers at 35 days of age
BW -Body weight; BWG -Body weight gain; FI -Feed intake; FCR -Feed conversion ratio; a,b Means followed by different letters in the same line differ significantly Tukey test, P<0.05.A, B Means followed by different letters in the same collum differ significantly Tukey test, P<0.05.

Table 9 : Effect of interaction between the supplementation sources of broilers at 42 days of age
BW -Body weight; BWG -Body weight gain; FI -Feed intake; FCR -Feed conversion ratio; a,b Means followed by different letters in the same line differ significantly Tukey test, P<0.05.A, B Means followed by different letters in the same column differ significantly Tukey test, P<0.05.

Table 11 : Effect of interaction between the supplementation sources of broilers at 42 days of age
Means followed by different letters in the same line differ significantly Tukey test, P<0.05.A, B Means followed by different letters in the same collum differ significantly Tukey test, P<0.05.

Table 13 : Effect of interaction between supplementation sources of broilers at 42 days of age
Means followed by different letters in the same line differ significantly Tukey test, P<0.05.A, B Means followed by different letters in the same column differ significantly Tukey test, P<0.05.Findings from this trial suggest NCA outperformed other sources evaluated.Aside from superior performance, NCA was also able to provide satisfactory results at lower doses.To support use recommendations, economic estimates accounting for the effects of additive and dose on production were presented in Table14(Investment per kg of feed per phase and weighted average in days) and Table15(Relative Gross Income calculation).